Real-time fluorescence measurement of cell-free endosome fusion: Regulation by second messengers

Abstract

A quantitative real-time assay of cell-free endosomal vesicle fusion was developed and applied to study fusion mechanisms in endosomes from baby hamster kidney (BHK-21) cells. The assay is based on an irreversible ~10- fold increase in BODIPY-avidin fluorescence on binding of biotinylated conjugates. BODIPY-avidin and biotin-dextran were internalized for 10 min at 37°C into separate populations of BHK-21 cells, and endosome fractions were prepared. Postnuclear supernatant fractions underwent ATP- and temperature- dependent fusion, as measured in a sensitive custom-built microfluorimeter by the continuous increase in BODIPY-avidin fluorescence. Fusion processes of efficiency >2.5% could be detected with 200-ms time resolution in sample volumes of 50 μL containing endosomes derived from ~4 x 104 cells. The fusion time course consisted of a distinct lag phase (up to 10 min) in which little fusion occurred, followed by an approximately exponential rise (t(1/2) 10-30 min; fusion efficiency ~15%). The lag phase was reduced by preincubation of separate endosome fractions with ATP at 37°C and by coincubation of endosomes at 22°C before the assay, suggesting a rate- limiting step involving binding of a soluble protein to the endosome membrane. Endosome fusion was strongly inhibited by GTPγS, N-ethylmaleimide, and AlF4. Endosome fusion was not affected by phorbol myristate acetate but was significantly inhibited by cAMP and bovine brain calmodulin. The results establish a sensitive real-time fluorescence assay to quantify the kinetics and extent of endosome fusion in a cell-free system and demonstrate regulation of early endosome fusion by cytosolic second messengers.